PROJECT SUMMARY Understanding the molecular basis of anatomical variation is a fundamental challenge in biology. In some cases, the genes that control anatomical defects in humans underlie normal variation in other species; therefore, a comprehensive understanding of the general molecular mechanisms of diversity promises a greater understanding of human health. I have pioneered molecular developmental and genetic studies of domestic pigeons as a model for dramatic anatomical variation. In just a few years, we have made rapid progress to discover the molecular underpinnings of complex traits in pigeons, including the discovery that genes underlying hereditary disease and cancer in humans also play key roles in animal diversity. This project seeks to deepen and broader our understanding of the molecular basis of typical and abnormal variation. The pigeon is an ideal system in which to pursue these goals because it features tremendous morphological variation within a single species, thereby facilitating genome-wide association studies, traditional genetic mapping, and functional developmental biology. First, we will identify the regulatory mechanisms that control forelimb and hindlimb identity. In certain breeds of domestic pigeon, our genetic mapping and developmental studies show that regulatory changes in two genes are associated with the replacement of scales by feathers on the feet. In humans, mutations in these same genes cause striking limb malformations. We will identify specific mutations causing regulatory changes in pigeons by testing enhancer constructs in ovo, and use high-throughput RNA sequencing to identity the downstream gene regulatory networks that control limb identity. Second, we will map the genes controlling major changes in craniofacial size and shape through genome-wide association scans and genetic mapping in laboratory crosses. We will use functional testing of candidate genes and transcriptome profiling to identify the molecular basis of radical variation in beak structures. The craniofacial skeleton of pigeons shows spectacular variation among breeds, and abnormal development of these same structures accounts for one-third of human birth defects. Therefore, understanding the molecular basis of this variation is critical to understanding of both natural variation and pathogenesis of human craniofacial disorders. Third, two classical pigeon mutants exhibit variation in both pigmentation and eye development. Phenomenological links are well established between pigment variation and eye development, but mechanistic links are often ambiguous. We have identified strong candidate genes for both mutants, and will use pigeons and other canonical model organisms to functionally test the impact of their altered expression. Together, these complementary genetic, genomic, and developmental approaches will identify the molecular basis of astonishing variation in an innovative model system, thereby opening new avenues to understand the conserved roles of specific genes in normal and disease variation among vertebrates.